Method for purifying single-stranded dna

Abstract

A method for purifying single-stranded DNA is provided. The method can comprise: mixing a sample containing single-stranded DNA with a single-stranded DNA binding protein, and incubating for a first time period to obtain a first product, the first product containing a conjugate of the single-stranded DNA and the single-stranded DNA binding protein; purifying the first product by using affinity chromatography to obtain a second product; and purifying the second product to at least remove the single-stranded DNA binding protein to obtain a target product containing a purified single-stranded DNA. Also provided are a kit for purifying single-stranded DNA, the kit comprising a single-stranded DNA binding protein and a purification reagent; and a use of a single-stranded DNA binding protein in purifying single-stranded DNA.

We claim: 
     
         1 . A method for purifying single-stranded DNA, characterized in that the method comprises:
 mixing a sample containing the single-stranded DNA with a single-stranded DNA binding protein, and incubating for a first time period to obtain a first product, wherein the first product contains a conjugate of the single-stranded DNA and the single-stranded DNA binding protein; and   purifying the first product to obtain a target product containing purified single-stranded DNA.   
     
     
         2 . The method according to  claim 1 , characterized in that when the sample is mixed with the single-stranded DNA binding protein, the single-stranded DNA binding protein has a final concentration of 0.5 ng/μL to 500 ng/μL. 
     
     
         3 . The method according to  claim 1 , characterized in that when the sample is mixed with the single-stranded DNA binding protein, the single-stranded DNA binding protein has a final concentration of 1 ng/μL to 200 ng/μL. 
     
     
         4 . The method according to  claim 1 , characterized in that the first time period is 30 minutes to 12 hours. 
     
     
         5 . The method according to  claim 1 , characterized in that the first time period is 2-4 hours. 
     
     
         6 . The method according to  claim 1 , characterized in that the sample is mixed with the single-stranded DNA binding protein and incubated at 30-37° C. 
     
     
         7 . The method according to  claim 1 , characterized in that the step of purifying the first product comprises:
 separating and purifying the first product to obtain a second product; and   purifying the second product to at least remove the single-stranded DNA binding protein, so as to obtain the target product containing the purified single-stranded DNA.   
     
     
         8 . The method according to  claim 7 , characterized in that the first product is separated and purified by affinity chromatography, ion exchange chromatography or gel filtration chromatography. 
     
     
         9 . The method according to  claim 8 , characterized in that the first product is separated and purified by the affinity chromatography. 
     
     
         10 . The method according to  claim 9 , characterized in that the single-stranded DNA binding protein carries a purification tag. 
     
     
         11 . The method according to  claim 10 , characterized in that the purification tag is a polyhistidine tag, a glutathione-S-transferase tag, a FLAG tag or a Strep-tagII tag. 
     
     
         12 . The method according to  claim 7 , characterized in that the second product is purified by a process selected from at least one of the following: phenol chloroform extraction, DNA adsorption spin column recovery, plasmid extraction ion exchange column recovery, isopropanol precipitation, ethanol precipitation and molecular sieve chromatography. 
     
     
         13 . The method according to  claim 1 , further comprising performing PCR amplification and DNA sequencing on the target product to verify whether the target product contains a complete sequence of the single-stranded DNA. 
     
     
         14 . The method according to  claim 1 , characterized in that the single-stranded DNA binding protein comprises a single-stranded DNA binding protein derived from  Escherichia coli , phage phi29,  Salmonella  phage Vi II-E1,  Thermus thermophilus  HB8,  Klebsiella  phage Phi K02,  Yersinia  phage Py54 or  Synechococcus  phage S-CBS2, a core functional fragment thereof, a homolog sequence thereof, or a mutant sequence thereof. 
     
     
         15 . The method according to  claim 14 , characterized in that the single-stranded DNA binding protein comprises a sequence that has at least 80% sequence identity with an amino acid sequence set forth in SEQ ID NO: 2, 9, 10 or 11 and has a function of binding to single-stranded DNA. 
     
     
         16 . A kit for purifying single-stranded DNA, characterized in that the kit comprises: a single-stranded DNA binding protein and a purification reagent. 
     
     
         17 . The kit according to  claim 16 , characterized in that the purification reagent is an affinity chromatography purification reagent. 
     
     
         18 . A single-stranded DNA binding protein for use in purifying single-stranded DNA.